PROJECT SUMMARY The molecular mechanisms through which cells sense nutrients remain largely unknown, but their elucidation is key to our understanding of metabolic regulation both in normal and disease states. At the center of nutrient sensing and growth regulation is an ancient protein kinase known as the mechanistic Target of Rapamycin Complex 1 (mTORC1). In response to the combined action of metabolic inputs such as nutrients, growth factors, energy and oxygen, mTORC1 translocates from the cytoplasm to the surface of lysosomes, where it becomes activated. Accumulating evidence indicates that aberrant mTORC1 activation at the lysosome could be a driving force in diseases ranging from cancer to type-2 diabetes to neurodegeneration. Lysosomal translocation and activation of mTORC1 requires the heterodimeric Rag guanosine triphosphatases (GTPases), which together with the pentameric Ragulator complex, form a nutrient-regulated scaffolding complex that physically anchors mTORC1 to the lysosomal surface. Combining dynamic imaging in cells with biochemical reconstitution and structural approaches, we recently discovered that the Ragulator-Rag complex is not static but is rather actively remodeled by nutrients, leading to spatial cycling of the Rag GTPases between the lysosomal surface and the cytoplasm. In turn, Rag cycling places a limit on the efficiency of mTORC1 capture and may facilitate its inactivation when nutrient levels fall. Importantly, Rag cycling is altered by cancer-specific mutations that affect mTORC1 signaling. Based on these findings, we hypothesize that spatial-temporal regulation of mTORC1 scaffolding is a novel and unrecognized mechanism to modulate the potency and selectivity of mTORC1 signaling responses, and that its disruption may drive the aberrant growth of mTORC1-driven cancers, including renal cell carcinoma and lymphoma. We will test this hypothesis via two highly complementary and innovative research aims. First, we will employ structure-guided mutagenesis to dissect the mechanisms that govern the assembly of the mTORC1- scaffolding complex in response to changing nutrient inputs. Second, we will characterize the mechanism of action of new-generation compounds we recently discovered, which block the assembly of the lysosomal mTORC1 scaffolding complex, and determine their ability to inhibit the metabolism and growth of mTORC1- driven cancers. Collectively, the proposed studies will generate new knowledge on the spatial-temporal regulation of mTORC1 signaling, and point the way to novel strategies to manipulate mTORC1 signaling in both normal and disease states.